Method for enumeration of mammalian cell micronuclei

ABSTRACT

The present invention relates a method for the enumeration of mammalian cell micronuclei, while distinguishing micronuclei from the chromatin of dead and dying cells. The method utilizes differential staining of chromatin from dead and dying cells, to distinguish the chromatin from micronuclei and nuclei that can be detected based upon fluorescent emission and light scatter following exposure to an excitatory light source. Counting of micronuclei events relative to the number of nuclei can be used to assess the DNA-damaging potential of a chemical agent, the DNA-damaging potential of a physical agent, the effects of an agent which can modify endogenously-induced DNA damage, and the effects of an agent which can modify exogenously-induced DNA damage. Kits for practicing the invention are also disclosed.

This application is a continuation of U.S. patent application Ser. No.12/244,179 filed Oct. 2, 2008, now U.S. Pat. No. 7,645,593, which is adivisional of U.S. patent application Ser. No. 11/166,433 filed Jun. 24,2005, now U.S. Pat. No. 7,445,910, which claims priority benefit of U.S.Provisional Patent Application Ser. No. 60/584,555 filed Jul. 1, 2004,the entire disclosure of which is hereby incorporated by reference inits entirety.

The present invention was made with funding received from the NationalCancer Institute under grants R43CA094493 and R44CA094493. The U.S.government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The induction of DNA damage and the resulting sequelae of mutations andchromosomal rearrangements are primary mechanisms by which cancersarise. These types of events have also been implicated in diseases suchas atherosclerosis, processes such as aging, and the development ofbirth defects such as Down syndrome. Therefore, there is an importantneed for sensitive methods which are capable of identifying chemical orphysical agents that can alter DNA. Given the tremendous cost oflong-term chronic studies such as 2-year carcinogenicity tests, short-and medium-term systems for predicting DNA damage potential continueplay a vital role in tumorigenic agent identification. In fact, the needfor short-term tests that have a high throughput capacity has never beengreater. Advances in molecular biology and combinatorial chemistry haveprovided large numbers of potential targets and many novel compoundsthat may be useful for treating or preventing disease. However, beforesuch agents can be tested and widely administered, acceptable toxicityto critical organs must be demonstrated. In the area of environmentalhealth and safety, many natural and industrially manufactured compoundsand formulations have not been adequately evaluated for toxicity. Inboth arenas, traditional toxicity evaluations are labor intensive andrequire extensive use of in vivo assays. This situation offersopportunities for methods that are able to quickly and inexpensivelydetermine toxicological profiles of potential therapeutic drugs andenvironmental agents.

Micronuclei are formed upon cell division in cells with DNAdouble-strand break(s) or dysfunctional mitotic spindle apparatus. Basedon this detailed understanding of micronuclei origin, the rodent-basedmicronucleus test has become the most widely utilized in vivo system forevaluating the clastogenic and aneugenic potential of chemicals (Heddle,“A Rapid In Vivo Test for Chromosome Damage,” Mutat. Res. 18:187-190(1973); Schmid, “The Micronucleus Test,” Mutat. Res. 31:9-15 (1975);Hayashi et al., “In Vivo Rodent Erythrocyte Micronucleus Assay. II. SomeAspects of Protocol Design Including Repeated Treatments, IntegrationWith Toxicity Testing, and Automated Scoring,” Environ. Mol. Mutagen.35:234-252 (2000)). These rodent-based tests are most typicallyperformed as erythrocyte-based assays. Since erythroblast precursors area rapidly dividing cell population, and their nucleus is expelled a fewhours after the last mitosis, micronucleus-associated chromatin isparticularly simple to detect in reticulocytes and normochromaticerythrocytes given appropriate staining (e.g., acridine orange) (Hayashiet al., “An Application of Acridine Orange Fluorescent Staining to theMicronucleus Test,” Mutat. Res. 120:241-247 (1983)).

One of the short-term test systems that is believed to hold greatpromise as a rapid tool for screening drug candidates and otherchemicals for genotoxic activity is the in vitro micronucleus test.Analogous to the way in vivo erythrocyte-based micronucleus tests havebecome more common than in vivo chromosome aberration analyses, agrowing consensus has been forming that in vitro micronucleus assayscould largely replace in vitro chromosome aberration studies. While bothendpoints are capable of detecting agents that cause structural ornumerical chromosome aberrations, in vitro micronucleus formation istechnically easier to perform and score. The difficulty, however, isidentifying the procedures that can reliably achieve an in vitromicronucleus assay that can satisfy the need for both fast and accurateresults.

The in vitro micronucleus test demonstrates high concordance withchromosome aberration analyses, but it is executed more rapidly andrequires less technical expertise (Matsuoka et al., “Evaluation of theMicronucleus Test Using a Chinese Hamster Cell Line as an Alternative tothe Conventional In Vitro Chromosomal Aberration Test,” Mutat. Res.272:223-236 (1993); Miller et al., “Comparative Evaluation of the InVitro Micronucleus Test and the In Vitro Chromosome Aberration Test:Industrial Experience,” Mutat. Res. 392:45-59 (1997); Miller et al.,“Evaluation of the In Vitro Micronucleus Test as an Alternative to theIn Vitro Chromosome Aberration Assay: Position of the GUM Working Groupon the In Vitro Micronucleus Test,” Mutat. Res. 410:81-116 (1998)).These characteristics have led to its widespread use as an efficient andrelatively simple method to screen drug candidates and other testarticles for clastogenic and aneugenic potential (Nesslany et al., “AMicromethod for the In Vitro Micronucleus Assay,” Mutagenesis 14:403-410 (1999)). Furthermore, there have been concerted efforts toestablish robust protocols so that the in vitro micronucleus test canserve as a source of cytogenetic damage information for regulatorysubmission purposes in place of in vitro chromosome aberration results(Albertini et al., “Detailed Data on In Vitro MNT and In Vitro CA:Industrial Experience,” Mutat. Res. 392:187-208 (1997); von der Hude etal., “In Vitro Micronucleus Assay with Chinese Hamster V79 Cells—Resultsof a Collaborative Study with In Situ Exposure to 26 ChemicalSubstances,” Mutat. Res. 468:137-163 (2000); Garriott et al., “AProtocol for the In Vitro Micronucleus Test. I. Contributions to theDevelopment of a Protocol Suitable for Regulatory Submissions from anExamination of 16 Chemicals with Different Mechanisms of Action andDifferent Levels of Activity,” Mutat. Res. 517:123-134 (2002); Phelps etal., “A Protocol for the In Vitro Micronucleus Test. II. Contribution tothe Validation of a Protocol Suitable for Regulatory Submissions from anExamination of 10 Chemicals with Different Mechanisms of Action andDifferent Levels of Activity,” Mutat. Res. 521:103-112 (2002);Kirsch-Volders et al., “Report from the In Vitro Micronucleus AssayWorking Group,” Mutat. Res. 540:153-163 (2003)). In fact theseactivities have progressed to the point that draft guidelines have beenwritten by the Organisation for Economic Co-operation and Development(“OECD”) (“Draft Proposal for a New Guideline 487: In Vitro MicronucleusTest,” June 2004).

Given the growing enthusiasm for the in vitro micronucleus endpoint,numerous efforts to automate the scoring phase of the technique havebeen described in the literature—methods based on image analysis, laserscanning cytometry, and flow cytometry have all been reported (Nüsse etal., “Flow Cytometric Analysis of Micronuclei Found in Cells AfterIrradiation,” Cytometry 5:20-25 (1984); Schreiber et al., “An AutomatedFlow Cytometric Micronucleus Assay for Human Lymphocytes,” Int. J.Radiat. Biol. 62:695-709 (1992); Schreiber et al., “Multiparametric FlowCytometric Analysis of Radiation-Induced Micronuclei in Mammalian CellCultures,” Cytometry 13:90-102 (1992); Vral et al., “The In VitroCytoKinesis-Block Micronucleus Assay: A Detailed Description of anImproved Slide Preparation Technique for the Automated Detection ofMicronuclei in Human Lymphocytes,” Mutagenesis 9:439-443 (1994);Verhaegen et al., “Scoring of Radiation-Induced MicronucleiCytokineses-Blocked Human Lymphocytes by Automated Image Analysis,”Cytometry 17:119-127 (1994); Wicker et al., “Image Processing Algorithmsfor the Automated Micronucleus Assay in Binucleated Human Lymphocytes,”Cytometry 19:283-294 (1995); Wessels et al., “Flow cytometric Detectionof Micronuclei by Combined Staining of DNA and Membranes,” Cytometry19:201-208 (1995); Viaggi et al., “Flow Cytometric Analysis ofMicronuclei in the CD2+ Subpopulation of Human Lymphocytes Enriched byMagnetic Separation,” Int. J. Radiat. Biol. 67:193-202 (1995); Nüsse etal., “Flow Cytometric Analysis of Micronuclei In Cell Cultures and HumanLymphocytes: Advantages and Disadvantages,” Mutat. Res. 392:109-115(1997); Roman et al., “Evaluation of a New Procedure for the FlowCytometric Analysis of In Vitro, Chemically Induced Micronuclei in V79Cells,” Environ. Molec. Mutagen. 32:387-396 (1998)). The mostestablished technique for high throughput in vitro micronuclei scoring,both in terms of years since original description and the number ofpeer-reviewed publications, is the flow cytometric (“FCM”) proceduredeveloped by Nüsse and colleagues (Nüsse et al., “Flow CytometricAnalysis of Micronuclei Found in Cells After Irradiation,” Cytometry5:20-25 (1984); Schreiber et al., “An Automated Flow CytometricMicronucleus Assay for Human Lymphocytes,” Int. J. Radiat. Biol.62:695-709 (1992); Schreiber et al., “Multiparametric Flow CytometricAnalysis of Radiation-Induced Micronuclei in Mammalian Cell Cultures,”Cytometry 13:90-102 (1992); Nüsse et al., “Flow Cytometric Analysis ofMicronuclei in Cell Cultures and Human Lymphocytes Advantages andDisadvantages,” Mutat. Res. 392:109-115 (1997)).

As the major limitation of FCM-based techniques has been their inabilityto distinguish true micronuclei from apoptotic bodies, methods fordifferential staining of micronuclei from the chromatin of dead anddying cells are needed.

The present invention overcomes the disadvantages of prior artapproaches, and satisfies the need of establishing a robust, reliable,high throughput in vitro micronucleus assay.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method for theenumeration of mammalian cell micronuclei, while distinguishingmicronuclei from the chromatin of dead and dying cells. This methodinvolves contacting a sample containing mammalian cells with a firstfluorescent DNA dye that permeates dead and dying cells but not viablecells, that covalently binds chromatin, and that has a fluorescenceemission spectrum. The sample is contacted with one or more lysissolutions that result in digestion of mammalian cell outer membranes butretention of nuclear membranes, thereby forming free nuclei and/ormicronuclei. The free nuclei and/or micronuclei are contacted with RNaseto substantially degrade RNA. Cellular DNA is stained with a secondfluorescent DNA dye having a fluorescent emission spectrum which doesnot substantially overlap with the fluorescent emission spectrum of thefirst fluorescent DNA dye. The first and second fluorescent DNA dyes areexcited with light of appropriate excitation wavelength. The fluorescentemission and light scatter produced by the nuclei and/or micronuclei aredetected, while chromatin from the dead and dying cells is excluded, andthe number of micronuclei in the sample relative to the number of nucleiis counted.

A second aspect of the present invention relates to a method ofassessing the DNA-damaging potential of a chemical or physical agent.This method involves exposing a sample containing mammalian cells to achemical or physical agent and performing the method according to thefirst aspect of the present invention. A significant deviation in thefrequency of micronuclei from a baseline micronuclei value in unexposedor vehicle control mammalian cells indicates the genotoxic potential ofthe chemical or physical agent.

A third aspect of the present invention relates to a method ofevaluating the effects of an agent which can modify endogenously-inducedDNA damage. This method of the present invention can be carried out byexposing mammalian cells to an agent that may modifyendogenously-induced genetic damage to mammalian cells. The methodaccording to the first aspect of the invention is performed with theexposed mammalian cells. A significant deviation in the frequency ofmicronuclei from a baseline micronuclei value in unexposed orvehicle-exposed mammalian cells indicates that the agent can modifyendogenous DNA damage.

A fourth aspect of the present invention relates to a method ofevaluating the effects of an agent which can modify exogenously-inducedDNA damage. This method of the present invention can be carried out byexposing mammalian cells to an exogenous agent that causes geneticdamage and an agent that may modify exogenously-induced genetic damage.The method according to the first aspect of the present invention isperformed with the exposed mammalian cells. A significant deviation inthe frequency of micronuclei from genotoxicant-exposed mammalian cellsindicates that the agent can modify exogenously-induced DNA damage.

A fifth aspect of the present invention relates to a kit that includes:one or more mammalian cell membrane lysis solutions; a first fluorescentDNA dye that permeates the dead and dying cells, but not viable cells; asecond fluorescent DNA dye having a fluorescent emission spectrum whichdoes not substantially overlap with a fluorescent emission spectrum ofthe first fluorescent DNA dye; and RNase A solution.

A sixth aspect of the present invention relates to a method of assessingthe cytotoxicity of a chemical or physical agent. This method involvesexposing mammalian cells to a chemical or physical agent and performingthe method according to the first aspect of the present invention. Asignificant deviation in the frequency of chromatin from dead and dyingcells from a baseline value in unexposed or vehicle control mammaliancells indicates the cytotoxic potential of the chemical or physicalagent.

A seventh aspect of the present invention relates to a method ofassessing the effect of a chemical or physical agent on the cell-cycleof mammalian cells. This method involves exposing mammalian cells to achemical or physical agent and performing the method according to thefirst aspect of the present invention. The detected nuclei are thendisplayed as a linear mode histogram. Dose-dependent perturbations arethen detected, which indicate an adverse effect of the chemical orphysical agent on the cell-cycle of mammalian cells.

The methods described herein provide for the enumeration of mammaliancell micronuclei using, preferably, flow cytometry technology. Theprimary advantage of this methodology relative to other flowcytometry-based procedures which have been reported to date is the useof a sequential staining procedure capable of differentially stainingmicronuclei and the chromatin of dead and dying cells, thus providingmore accurate and reliable micronuclei measurements (see Nüsse et al.,“Flow Cytometric Analysis of Micronuclei Found in Cells AfterIrradiation,” Cytometry 5:20-25 (1984); Nüsse et al., “FactorsInfluencing the DNA Content of Radiation-Induced Micronuclei,” Int. J.Radiat. Biol. 62:587-602 (1992); and Nüsse et al., “Flow CytometricAnalysis of Micronuclei in Cell Cultures and Human LymphocytesAdvantages and Disadvantages,” Mutat. Res. 392:109-115 (1997), which arehereby incorporated by reference in their entirety). Thus, the presentinvention identifies procedures that can be employed for an automated invitro micronucleus assay that can be used to evaluate agents (e.g.,chemical or physical agents) for toxicity to mammalian cells. Theprocedure is fast, reliable, and accurate, and can be performed withoutthe need for dosing of animals. Consequently, significant cost savingscan be afforded by the present invention in the process of testingagents for geno- and/or cytotoxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of an exemplary cell stainingtechnique according to the methods of the present invention. This twodye, sequential staining procedure enhances the reliability of flowcytometry-based analyses by differentially staining micronuclei andchromatin associated with dead and dying cells. Ethidium monoazide(“EMA”) represents a preferred first DNA dye, and SYTOX represents apreferred second DNA dye.

FIGS. 2A-H are histogram and bivariate plots of L5178Y cells treatedwith vehicle (FIGS. 2A-G) or 20 μg methyl methanesulfonate/ml (FIG. 2H).These graphs illustrate the gating strategy used to discriminatemicronuclei from apoptotic chromatin and other spurious events. Forevents to be displayed and scored by FIGS. 2G and 2H, they needed tomeet each of the following six criteria: within a side scatter versusforward scatter region (FIG. 2A); at least 1/100 the SYTOX-associatedfluorescence as G1 nuclei (FIG. 2B); within a region that excludesdoublets (FIG. 2C); within a forward scatter versus SYTOX fluorescenceregion (FIG. 2D); within a side scatter versus SYTOX fluorescence region(FIG. 2E); and EMA-negative (FIG. 2F). The position of the micronucleusscoring region (MN) was designed to score events that exhibited 1/100 to1/10 the SYTOX fluorescence intensity of G1 nuclei.

FIG. 3 is a graph showing apoptosis resulting from 1 hr heat treatment,as detected by four different staining techniques. These data illustratethe rates at which dying cells acquire these different stainingcharacteristics. EMA is seen to be a relatively early indicator ofapoptosis, as it rivals YO-PRO-1, a dye which is known to stain earlystage apoptotic cells.

FIG. 4 is a graph showing micronuclei frequencies plotted againstculture conditions. The “Healthy Culture” consisted of log phase L5178Ycells, whereas the “Apoptotic Culture” consisted of cells which wereheat-treated. These cultures were combined, then processed for flowcytometric scoring. When the EMA staining criterion was not employed(white bars), the frequency of micronuclei was artificially high due tocontamination of dead and dying cells' chromatin. On the other hand, thefrequency of micronuclei was not appreciably affected when an EMAstaining criterion was used to exclude chromatin from dead and dyingcells (black bars).

FIGS. 5A-M are graphs showing genotoxicity and cytotoxicity measurementsfor L5178Y cells treated with each of 9 chemicals. The Y-axis depictsthe mean frequency of micronuclei obtained by microscopic inspection(black bars, 2000 cells scored per culture) and via flow cytometric(FCM) analysis (white bars, mean of 3 measurements per culture, with SEMbars). An index of cytotoxicity (% relative survival) is displayed onthe YY-axis. Cells were treated for 4 hrs and harvested for analysisafter an additional 20 hr recovery for experiments depicted by FIGS. 5A,5B, 5C, 5D, 5E, 5F, 5H, 5J, and 5L. Treatment was continuous (24 hrs, norecovery) for experiments depicted in FIGS. 5G, 5I, 5K, and 5M.

FIGS. 6A-C are graphs showing SYTOX-associated fluorescence (FL1-A)plotted for L5178Y cells treated with 0 (FIG. 6A), 10 (FIG. 6B), and 20(FIG. 6C) μg methyl methanesulfonate (MMS) per ml. These data providecell cycle information that is acquired simultaneously with micronucleusdata. This example depicts a dose-dependent G2/M block.

FIGS. 7A-D are graphs showing EMA versus SYTOX-associated fluorescenceplotted for each of 4 dexamethasone-treated cultures (given 24 hrcontinuous treatment). The percent EMA-positive events is related tocell membrane integrity, and therefore provides supplementalcytotoxicity information that is acquired concurrently with flowcytometric micronucleus measurements.

FIG. 8 is a graph showing genotoxicity and cytotoxicity measurements forCHO-K1 cells treated with a range of methyl methanesulfonateconcentrations. The Y-axis depicts the mean frequency of micronucleiobtained by microscopic inspection (black bars, 2000 cells scored perculture) and via flow cytometric (FCM) analysis (white bars, mean of 3measurements per culture, with SEM bars). An index of cytotoxicity (%relative survival) is displayed on the YY-axis. Cells were treated for 4hrs and harvested for analysis after an additional 20 hr recovery.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for the enumeration ofmicronuclei in mammalian cells using a standard, widely available flowcytometer apparatus which provides for excitation of fluorochromes anddetection of resulting fluorescent emissions as well as light scattersignals.

A first aspect of the present invention relates to a method for theenumeration of mammalian cell micronuclei, while distinguishingmicronuclei from the chromatin of dead and dying cells. This methodinvolves contacting a sample containing mammalian cells with a firstfluorescent DNA dye that permeates dead and dying cells but not viablecells, that covalently binds chromatin, and that has a fluorescenceemission spectrum. The sample is contacted with one or more lysissolutions that result in digestion of mammalian cell outer membranes butretention of nuclear membranes, thereby forming free nuclei and/ormicronuclei. The free nuclei and/or micronuclei are contacted with RNaseto substantially degrade RNA. Cellular DNA is stained with a secondfluorescent DNA dye having a fluorescent emission spectrum that does notsubstantially overlap with the fluorescent emission spectrum of thefirst fluorescent DNA dye. The first and second fluorescent DNA dyes areexcited with light of appropriate excitation wavelength. The fluorescentemission and light scatter produced by the nuclei and/or micronuclei aredetected, while chromatin from the dead and dying cells is excluded, andthe number of micronuclei in the sample relative to the number of nucleiis counted.

Mammalian cells suitable for carrying out the methods of the presentinvention include, without limitation, immortalized cell lines, as wellas cells which have only recently been harvested from mammalian speciesand placed into culture (i.e., primary cell cultures).

Preferred primary cell cultures are those that divide in culture (i.e.,with appropriate growth media, which for some cell types requires theinclusion of cytokines and/or other factors such as mitogens). Exemplarycell types that can be screened easily using the methods of the presentinvention include, without limitation, blood lymphocytes, bonemarrow-derived stem cells, hepatocytes, and keratinocytes.

Exemplary immortalized cell lines, include, without limitation, L5178Ycells, CHO (Matsuoka et al., “Evaluation of the Micronucleus Test Usinga Chinese Hamster Cell Line as an Alternative to the Conventional InVitro Chromosomal Aberration Test,” Mutat. Res. 272:223-236 (1993);Garriott et al., “A Protocol for the In Vitro Micronucleus Test. I.Contributions to the Development of a Protocol Suitable for RegulatorySubmissions form an Examination of 16 Chemicals with DifferentMechanisms of Action and Different Levels of Activity,” Mutat. Res.517:123-134 (2002); Phelps et al., “A Protocol for the In VitroMicronucleus Test. II. Contributions to the Validation of a ProtocolSuitable for Regulatory Submissions from an Examination of 10 Chemicalswith Different Mechanisms of Action and Different Levels of Activity,”Mutat. Res. 521:103-112 (2002); which are hereby incorporated byreference in their entirety) V79 (von der Hude et al., “In VitroMicronucleus Assay with Chinese Hamster V79 Cells—Results of aCollaborative Study with In Situ Exposure to 26 Chemical Substances,”Mutat. Res. 468:137-163 (2000), which is hereby incorporated byreference in its entirety), and TK6 (Zhan et al., Genotoxicity ofMicrocystin-LR in Human Lymphoblastoid TK6 Cells,” Mutat. Res. 557:1-6(2004), which is hereby incorporated by reference in its entirety). Eachof these cell types is widely used in genotoxicity investigations. Thecharacteristic that is most likely to define the compatibility of thisscoring system with any particular cell line will be the kinetics bywhich apoptosing cells become permeable to the first dye. It ispreferable that permeability occurs before nuclear fragmentation takesplace in order to effectively exclude apoptotic bodies from themicronuclei scoring region. As noted in the Examples, cells that grow asa suspension culture (i.e., L5178Y) and cells that grow as an attachmentculture (i.e., CHO-K1) have been used with success.

Micronuclei are membrane-bound, extra-nuclear, sub-2n DNA structuresresulting from double-strand chromosome breaks or from the dysfunctionof mitotic spindle apparatus. Micronuclei are also known as Howell-Jollybodies in the hematology literature.

Chromatin of dead and dying cells is DNA derived from cells which are nolonger viable, or from cells which have progressed to an irreversiblestage of cell death. Thus, “dead and dying cells” is meant to encompassnecrotic cell death typified by cytoplasmic swelling and rupture, aswell as apoptotic cell death which is usually characterized by cellularand nuclear shrinkage, condensation of chromatin, and fragmentation ofnuclei.

This first fluorescent DNA dye can be any such dye that can permeate thedead and dying cells, and then covalently bind chromatin. Preferably,the first fluorescent DNA dye is, at the time of contacting the cells inculture, in an inactive form. Thereafter, the dye is activated to areactive form, which is controlled by conditions that can be easilymanipulated in a laboratory setting (e.g., by light activation, changein pH, etc.). Upon activation, the dye should bind covalently to DNA,i.e., chromatin. When the dye is covalently bound to the DNA of dead anddying cells, it changes the nature of staining away from an equilibriumsituation. In particular, this approach for staining ensures that thefluorescent signal that is imparted to dead and dying cells is notdiminished during subsequent cell processing steps. In a preferredembodiment, the first fluorescent DNA dye is ethidium monoazide (“EMA”),which is efficiently converted to a reactive form throughphotoactivation.

The one or more lysis solutions can be any suitable lysis solution, orcombination thereof, for cell membrane lysis. According to oneembodiment, first and second lysis solutions are provided, with thefirst lysis solution having NaCl, Na-Citrate, and IPGAL in deionizedwater and the second lysis solution having citric acid and sucrose indeionized water. Cell lysis preferably occurs according to modificationsto a procedure that has been described in the literature (Nüsse et al.,“Flow Cytometric Analysis of Micronuclei Found in Cells AfterIrradiation,” Cytometry 5:20-25 (1984); Nüsse et al., “FactorsInfluencing the DNA Content of Radiation-Induced Micronuclei,” Int. J.Radiat. Biol. 62:587-602 (1992); and Nüsse et al., “Flow CytometricAnalysis of Micronuclei in Cell Cultures and Human Lymphocytes:Advantages and Disadvantages,” Mutat. Res. 392:109-115 (1997), which arehereby incorporated by reference in their entirety).

In one embodiment of the methods of the present invention, contactingthe sample with one or more lysis solutions and contacting the freenuclei and/or micronuclei with RNase may be carried out simultaneously.Alternatively, these steps are carried out sequentially.

In an another embodiment, contacting the sample with one or more lysissolutions, contacting the free nuclei and/or micronuclei with RNase, andstaining cellular DNA with a second fluorescent DNA dye are carried outsimultaneously. Alternatively, these steps are carried out sequentially.

Suitable second fluorescent DNA dyes are capable of staining cellularDNA at a concentration range detectable by flow cytometry, and have afluorescent emission spectrum that does not substantially overlap withthe fluorescent emission spectrum of the first fluorescent DNA dye. Itshould be appreciated by those of ordinary skill in the art that othernucleic acid dyes are known and are continually being identified. Anysuitable nucleic acid dye with appropriate excitation and emissionspectra can be employed, such as YO-PRO-1, SYTOX Green, SYBR Green I,SYTO11, SYTO12, SYTO13, BOBO, YOYO, and TOTO. A preferred secondfluorescent DNA dye is SYTOX Green.

The first and second fluorescent DNA dyes have sufficiently distinctemission maxima. Preferably, the first and second fluorescent DNA dyeshave similar excitation spectra. The advantage of similar excitationspectra is that it affords the use of the more widespread single-laserflow cytometer (as opposed to dual laser flow cyotometers). For example,when the first fluorescent DNA dye is EMA and the second fluorescent DNAdye is SYTOX Green, both the first and second fluorescent dyes aresufficiently excited by a flow cytometer equipped with a single 488 nmlaser, while their different emission maxima can be detected by twoseparate detectors using standard filter sets (FIG. 1).

Single-laser flow cytometric analysis uses a single focused laser beamwith an appropriate emission band to excite the first and secondfluorescent DNA dyes. As stained nuclei, micronuclei, and chromatindebris pass through the focused laser beam, they exhibit a fluorescentemission maxima characteristic of the fluorescent dye(s) associatedtherewith. Dual- or multiple-laser flow cytometric analysis uses two ormore focused laser beams with appropriate emission bands in much thesame manner as described for the single-laser flow cytometer. Differentemission bands afforded by the two or more lasers allow for additionalcombinations of nucleic acid dye(s) to be employed.

Preferably, the flow cytometer is equipped with appropriate detectiondevices to enable detection of the fluorescent emissions and lightscatter produced by the nuclei, micronuclei, and chromatin debris. These“light scatter” signals serve as additional criteria which helpsdiscriminate nuclei and micronuclei from apoptotic chromatin and otherdebris. The use of light scatter parameters to serve as additionalcriteria for accurately measuring micronuclei by flow cytometry has beendescribed in the literature (Nüsse et al., “Flow Cytometric Analysis ofMicronuclei in Cell Cultures and Human Lymphocytes: Advantages andDisadvantages,” Mutat. Res. 392:109-115 (1997), which is herebyincorporated by reference in its entirety), which is demonstrated inconjunction with an ethidium monoazide-labeling criterion in theexamples below.

A further aspect of the present invention relates to a method ofassessing the DNA-damaging potential of a chemical or physical agent.This method involves exposing a sample containing mammalian cells to achemical or physical agent and performing the method according to thefirst aspect of the present invention. A significant deviation in thefrequency of micronuclei from a baseline micronuclei value in unexposedor vehicle control mammalian cells indicates the genotoxic potential ofthe chemical or physical agent.

Physical agents which are known to damage DNA include, withoutlimitation, ionizing radiation, such as gamma and beta radiation, and UVradiation.

Chemical agents which are known to damage DNA include, withoutlimitation, inorganic genotoxicants (e.g., arsenic, cadmium and nickel),organic genotoxicants (especially those used as antineoplastic drugs,such as cyclophosphamide, cisplatin, vinblastine, cytosine arabinoside,and others), anti-metabolites (especially those used as antineoplasticdrugs, such as methotrexate and 5-fluorouracil), organic genotoxicantsthat are generated by combustion processes (e.g., polycyclic aromatichydrocarbons such as benzo(a)pyrene), as well as organic genotoxicantsthat are found in nature (e.g., aflatoxins such as aflatoxin B1).

The methods of the present invention are suitable for assessing theDNA-damaging potential of both physical and chemical agents, eitheralone or in combination with other such agents. Agents which have knownDNA-damaging potential and agents which are not known to haveDNA-damaging potential or are presumed not to have DNA-damagingpotential can be tested. In particular, physical and chemical agentswhich are under current investigation for therapeutic treatment, oragents which are being screened for potential therapeutic treatment areamenable to the methods of the present invention.

In carrying out the methods of the present invention, exposure ofmammalian cells to physical or chemical agents is preferably carried outfor a predetermined period of exposure time. Preferred exposure time fordetecting chromosome breaking (i.e., clastogenic) agents is betweenabout 3 and about 24 hours. There are some reports which suggest that apreferred exposure time for detecting aneugenic agents is approximately24 hours (Phelps et al., “A Protocol for the In Vitro Micronucleus Test.II. Contributions to the Validation of a Protocol Suitable forRegulatory Submissions from an Examination of 10 Chemicals withDifferent Mechanisms of Action and Different Levels of Activity,” Mutat.Res. 521:103-112 (2002), which is hereby incorporated by reference inits entirety).

Methods of assessing the DNA-damaging potential of a physical orchemical agent may further involve a delay between the end of exposureand prior to performing cell harvest, staining, membrane lysis, and flowcytometric analysis according to the first aspect of the presentinvention. When employed, the delay or “recovery” period is preferablybetween about 5 minutes to about 24 hours, although longer or shorterdelays can also be utilized.

To some degree, exposure time and recovery periods will be cell line-and chemical class-dependent. Persons of skill in the art can readilyoptimize the methods of the present invention for different cell linesand different physical or chemical agents.

Certain agents may offer protection from DNA damage, while othersmagnify risk of damage. The present invention can be used to evaluatethe effects of an agent which can modify (i.e., enhance or suppress)such damage. To assess the suspected protective effects of an agent, itcan be added to the culture of cells prior to, concurrently with, orsoon after addition of a known genotoxicant. Any protective effectafforded by the agent can be measured relative to damage caused by thegenotoxicant agent alone. For example, putative protective agents can bevitamins, bioflavonoids and anti-oxidants, dietary supplements (e.g.,herbal supplements), or any other protective agent, naturally occurringor synthesized by man.

To assess the ability of an agent to synergistically or additivelyenhance genotoxicity, the agent can be added to the culture of cellsprior to, concurrently with, or shortly after addition of a knowngenotoxicant. Any additive or synergistic effect caused by the agent canbe measured relative to damage caused by the genotoxicant agent alone.

Concurrent cytotoxicity assessment of chemical and/or physical agents(with or without protective agents or enhancing agents) can also bemade, pursuant to the methods of the present invention, such as (i) cellcycle effects based on the fluorescence intensity of the secondfluorescent DNA dye which is exhibited by nuclei and (ii) cytotoxicitybased on the percentage of particles that exhibit fluorescenceassociated with the first fluorescent DNA dye. The former can beachieved by assessing histograms to determine cell cycle progression ofthe cell population.

Thus, another aspect of the present invention relates to a method ofassessing the cytotoxicity of a chemical or physical agent. This methodinvolves exposing mammalian cells to a chemical or physical agent andperforming the method according to the first aspect of the presentinvention. A significant deviation in the frequency of chromatin fromdead and dying cells from a baseline value in unexposed or vehiclecontrol mammalian cells indicates the cytotoxic potential of thechemical or physical agent.

A further aspect of the present invention relates to a method ofassessing the effect of a chemical or physical agent on the cell-cycleof mammalian cells. This method involves exposing mammalian cells to achemical or physical agent and performing the method according to thefirst aspect of the present invention. The detected nuclei are thendisplayed as a linear mode histogram. Dose-dependent perturbations arethen detected, which indicate an adverse effect of the chemical orphysical agent on the cell-cycle of mammalian cells.

Another aspect of the present invention relates to a method ofevaluating the effects of an agent which can modify endogenously-inducedDNA damage. This method of the present invention can be carried out byexposing mammalian cells to an agent that may modifyendogenously-induced genetic damage to mammalian cells. The methodaccording to the first aspect of the invention is then performed withthe exposed mammalian cells. A significant deviation in the frequency ofmicronuclei from a baseline micronuclei value in unexposed orvehicle-exposed mammalian cells indicates that the agent can modifyendogenous DNA damage.

A further aspect of the present invention relates to a method ofevaluating the effects of an agent which can modify exogenously-inducedDNA damage. This method of the present invention can be carried out byexposing mammalian cells to an exogenous agent that causes geneticdamage and an agent that may modify exogenously-induced genetic damage.The method according to the first aspect of the present invention isthen performed with the exposed mammalian cells. A significant deviationin the frequency of micronuclei from genotoxicant-exposed mammaliancells indicates that the agent can modify exogenously-induced DNAdamage.

Yet another aspect of the present invention relates to a kit thatincludes: one or more mammalian cell membrane lysis solutions; first andsecond fluorescent DNA dyes as described above; and RNase A solution.

The kit may also include instructions that describe cell harvest andstaining procedures and micronucleus scoring via flow cytometry. The kitmay also include a computer readable storage medium that contains acytometry data acquisition template for flow cytometric micronucleusscoring. A container having an in vitro culture of mammalian cells mayalso be included in the kit of the present invention.

The importance and utility of this in vitro micronuclei scoring systemwill be magnified considerably by miniaturization and processautomation. Experiments described in the Examples, infra, were performedwith relatively large volumes of cells, and 1.5×10⁶ cells permeasurement were processed for each flow cytometry analysis. Thistranslates to high test article requirements and more culture medium,cells, etc., than is necessary to obtain reliable, repeatable results.For purposes of the present studies, the large number of cellsfacilitated parallel microscopy and several flow cytometry measurementsper flask. This design helped to evaluate the reproducibility of cellhandling, staining, and flow cytometry-analysis operations. However,significant reductions to numbers of cells and treatment volumes willallow for wide adoption of this or other techniques, especially in thecontext of lead prioritization and other early safety evaluationscreening goals. Lysis solution volumes and/or dye concentrations can beadjusted and optimized for miniaturization.

Screening programs that wish to evaluate large numbers of chemicals willbenefit from automation of processing steps. For instance, with flowcytometry, autoloaders (e.g., robotic equipment) are available which canoperate in “walk-away” mode. Multi-well plates for sequential analysisof high volumes of work and other large-scale automation tools such ascarousels are available to further automate the methods of the presentinvention. The processes described herein are expected to be fullycompatible with such systems.

The degree to which micronuclei events in mitogen-stimulated lymphocytescan be more easily and rapidly scored relative to chromosome aberrationsis in large part responsible for the cytokinesis-block micronuclei assayreplacing chromosome aberration analyses as the cytogenetic damageendpoint of choice for human biomonitoring and epidemiological studies(Bonassi et al., “Human Population Studies with Cytogenetic Biomarkers:Review of the Literature and Future Prospectives,” Environ. Molec.Mutagen. 45:258-270 (2005), which is hereby incorporated by reference inits entirety). Automation of the scoring phase of this process wouldsupply additional impetus to utilize the micronuclei endpoint inhuman-based studies, perhaps encouraging its use in studies and onscales that are not traditionally viewed as compatible with thisendpoint due to logistical and/or resource considerations.

EXAMPLES

The examples below are intended to exemplify the practice of the presentinvention but are by no means intended to limit the scope thereof.

Materials and Methods

Chemicals

The identities of the nine chemicals evaluated in the followingexamples, as well as solvent and other information, are listed in Table1.

TABLE 1 Chemicals Chemical Cas No. Solvent Genotoxic Mechanism Methylmethanesulfonate 66-27-3 PBS Alkylation Hydroxyurea 127-07-1 WaterRibonucleoside reductase inhibitor Etoposide 33419-42-0 DMSOTopoisomerase II inhibitor Cyclophosphamide monohydrate 6055-19-2 WaterDNA cross-linker Benzo[a]pyrene 50-32-8 DMSO Diol epoxides, reactive Ospecies Vinblastine sulfate 143-67-9 DMSO Mitotic spindle poison Sucrose57-50-1 RPMI + HS Non-genotoxicant Tributyltin methoxide 1067-52-3Ethanol Presumed non-genotoxicant Dexamethasone 50-02-2 DMSO Presumednon-genotoxicant PBS = phosphate buffered saline RPMI + HS = growthmedium DMSO = dimethyl sulfoxideEach of these nine chemicals were purchased from Sigma-Aldrich Corp.(St. Louis, Mo.). Dimethyl sulfoxide (CAS No. 67-68-5), ethanol (CAS No.64-17-5), DL-isocitric acid (CAS No. 1637-73-6), β-nicotinamide adeninedinucleotide phosphate sodium salt (CAS No. 1184-16-3), IGEPAL CA-630(CAS No. 9036-19-5), propidium iodide (CAS No. 25535-16-4), fluoresceindiacetate (CAS No. 596-09-8), acridine orange (CAS No. 65-61-2), sodiumcitrate (CAS No. 6132-04-3), citric acid (CAS No. 77-92-9), sucrose (CASNo. 57-50-1), and RNase A were also obtained from Sigma-Aldrich Corp.NaCl (CAS No. 7647-14-5) was purchased from J. T. Baker (Phillipsburg,N.J.). Annexin V-PE was obtained from the “Annexin V-PE ApoptosisDetection Kit I” (BD Pharmingen, La Jolla, Calif., cat no. 559763). Thefluorescent dyes ethidium monoazide (cat. no. E1374), SYTOX Green (cat.no. S7020) and YO-PRO-1 iodide (cat. no. Y3603), were obtained fromMolecular Probes, Eugene, Oreg.

Cells, Culture Medium, and Metabolic Activation

The L5178Y (tk+/−) and CHO-K1 cells used in these studies were fromAmerican Type Tissue Collection (ATCC) (Manassas, Va.). Cells weremaintained in culture medium at 37° C., 5% CO₂, and in a humidatmosphere. Cells were maintained between approximately 1×10⁴ and 1×10⁶cells/ml for routine passage.

For L5178Y cells, culture medium consisted of RPMI 1640 supplementedwith 200 mM L-glutamine, 100 IU penicillin and 100 μg/ml streptomycin,to which heat inactivated horse serum was added for 10% v/v finalconcentration (all from MediaTech Inc., Herndon, Va.). Serum free mediumwas identical to culture medium but without 10% horse serum.

For CHO-K1 cells, culture medium consisted of HAM F-12 supplemented with1.5 g/L sodium bicarbonate, 100 IU/ml penicillin and 100 μg/mlstreptomycin, to which heat inactivated fetal bovine serum was added for10% v/v final concentration (all from MediaTech Inc., Herndon, Va.).Serum free medium was identical to culture medium but without 10% fetalbovine serum. As CHO-K1 is an attachment cell line, routine passage andother instances of cell harvest required detachment of cells fromculture vessels. This was accomplished with 0.25% trypsin/2.21 mM EDTAsolution (from MediaTech Inc.).

The metabolic activation system consisted of 1 part rat liver S9post-mitochondrial fraction (Molecular Toxicology, Boone, N.C.) and 2.3parts cofactor mixture. The cofactor mixture was β-NADP at 20 mg/ml andDL-isocitric acid at 85 mg/ml dissolved in serum free medium (pHadjusted to approximately 7.2 with 1 N NaOH).

Heat Shock

Apoptosis was induced in log phase L5178Y cells by placing a flask in adry 47° C. oven for 1 hr. During this 1 hr period, the flask was tightlysealed. After this heat shock treatment, the culture was returned to a37° C. incubator with 5% CO₂, thereby allowing the apoptotic program toprogress. At 1 hr intervals, cells were harvested and incubated witheach of the following four reagents in order to evaluate the percentageof dead cells: YO-PRO-1 (Idziorek et al., “YOPRO-1 PermitsCytofluorometric Analysis of Programmed Cell Death (Apoptosis) WithoutInterfering With Cell Viability,” J. Immunological Methods 185:249-258(1995), which is hereby incorporated by reference in its entirety),Annexin-PE (Vermes et al., “A Novel Assay for Apoptosis. Flow CytometricDetection of Phosphatidylserine Expression on Early Apoptotic CellsUsing Fluorescein Labelled Annexin V,” J. Immunol. Meth. 184:39-51(1995), which is hereby incorporated by reference in its entirety),propidium iodide (Darzynkiewicz et al., “Cytometry in Cell Necrobiology:Analysis of Apoptosis and Accidental Cell Death (Necrosis),” Cytometry27:1-20 (1997), which is hereby incorporated by reference in itsentirety), and EMA (Riedy et al., “Use of a Photolabeling Technique toIdentify Nonviable Cells in Fixed Homologous or Heterologous CellPopulations,” Cytometry 12:133-139 (1991), which is hereby incorporatedby reference in its entirety). Flow cytometry analysis (488 nmexcitation) was performed and the percentage of fluorochrome-positivecells was calculated based on the acquisition of 20,000 total cells persample.

This same heat shock procedure was used in a subsequent experiment togenerate a culture with a high frequency of apoptotic cells (i.e., 1 hrheat treatment, 2 hrs of progression at 37° C.). The heat shockedculture was then added to healthy log phase cells at various ratios.These cultures were processed for flow cytometry scoring of micronucleiusing the EMA-SYTOX labeling method described infra.

Chemical Treatment and Cytotoxicity Measurements

L5178Y cells were treated over a range of chemical concentrations inculture medium, one 25 cm² flask per concentration. During treatment,cells were at 3×10⁵/ml in a volume of 20 ml per flask (this high numberof cells facilitated multiple readings per flask). After a 4 hrtreatment period, cells were washed one time via centrifugation at600×g, resuspended in a volume of 40 ml culture medium, and transferredto 75 cm² flasks. Cells were re-incubated at 37° C. for 20 additionalhrs—the equivalent of two normal doubling times. For treatments thatinvolved the promutagens benzo[a]pyrene and cyclophosphamide, the 4 hrtreatment period was conducted in the presence of 10% v/v metabolicactivation system.

L5178Y cells were also exposed to the chemicals sucrose, tributyltinmethoxide, dexamethasone, and vinblastine were also evaluated with a 24hr exposure protocol. In this case, cultures (1.5×10⁵ cells/ml, 40ml/flask) were continuously exposed to chemical over a range ofconcentrations. Cells were harvested after 24 hrs of incubation at 37°C. without recovery.

On the day before treatment, CHO-K1 cells were harvested and counted,and their density was adjusted to 6×10⁴/ml in culture medium. This cellsuspension was added to 75 cm² flasks (20 ml per flask; one flask perexposure group). After incubating for approximately 16 to 24 hrs at 37°C., spent culture medium was replaced with 20 ml fresh, pre-warmedmedium per flask. Methyl methanesulfonate was added from 1000× strengthstock solutions for final concentrations of 0, 10, 20, 30, and 40 μg/ml,at which time cultures where reincubated at 37° C. After a 4 hrtreatment period, culture medium was aspirated, cells were rinsed onetime with an isotonic salt solution, and then 20 ml fresh, pre-warmedculture medium was added to each flask. These cultures were reincubatedfor a 20 hr recovery period. At the time of cell harvest, 2.5 mltypsin/EDTA solution per flask was used to detach cells from culturevessels. Cells were collected via centrifugation and responded inculture medium.

Once L5178Y or CHO-K1 cells were collected as described above,cytotoxicity measurements were performed as follows: 0.5 ml of eachculture was added to flow cytometry tubes containing fluoresceindiacetate (“FDA”) (0.075 μg/ml), propidium iodide (“PI”) (25 μg/ml), and2.5 μm Carmine 580/620 microspheres (Molecular Probes, Eugene, Oreg.).The concentration of these “counting beads” was determined with ahemacytometer. This facilitated calculation of the absolute number oflive (FDA+) and dead (PI+) cells via flow cytometric analysis (Brando etal., “Cytofluorometric Methods for Assessing Absolute Numbers of CellSubsets in Blood,” Cytometry 42:327-346 (2000), which is herebyincorporated by reference in its entirety). Relative survival was thencalculated as follows: The number of FDA+ cells in each chemicaltreatment culture was expressed as a percentage of the number of FDA+cells in the concurrent solvent control culture. Only those treatedflasks that exhibited≦60% reduction to relative survival were preparedfor microscopy- and flow cytometric-based micronucleus analyses asdescribed infra. Sucrose was tested up to 5 mg/ml, as significantcytotoxicity was not observed.

Flow Cytometric Scoring of Micronuclei: Cell Harvest, Staining and Lysis

At the time of cell harvest, FDA+ and PI+ cell counts were performed asdescribed supra. For each culture which exhibited≦60% reduction to FDA+cells relative to solvent control, 1.5×10⁶ total cells were transferredto each of three 15 ml centrifuge tubes (to provide three independentlystained and flow cytometric-analyzed specimens per flask). Cells werecollected via centrifugation at approximately 600×g for 5 minutes.Supernatants were aspirated, and cells were resuspended with gentletapping. 200 μl “Buffer Solution” (PBS with 2% heat-inactivated fetalbovine serum, both from MediaTech) was added to each tube and cells werethen transferred to flow cytometry tubes (transparent polystyrene) with100 μl Nuclei Acid Dye 1 Solution (0.125 mg/ml EMA prepared in BufferSolution). These tubes were placed in racks and submerged to a depth ofapproximately 2 cm in crushed ice. A visible light source (60 watt lightbulb) was positioned approximately 30 cm above the tubes for 20 minutes.

After the photoactivation period, 800 μl cold buffer solution was addedto each sample. From this point forward, exposure of samples to lightwas minimized with dim lighting and foil. The contents of the tubes werethen transferred to 15 ml polypropylene centrifuge tubes, and 8 ml ofcold buffer solution was added to each sample. Cells were collected viacentrifugation, and supernatants aspirated such that approximately 50 μlof supernatant remained per tube. Cells were gently resuspended withtapping, and were maintained at room temperature until the followprocedures were initiated (within 30 minutes). 1 ml “Lysis Solution 1”was added slowly to each tube (approximately 15 seconds per sample).Lysis Solution 1 was prepared with deionized water and 0.584 mg/ml NaCl,1 mg/ml sodium citrate, 0.3 μl/ml IGEPAL, 1 mg/ml RNase A, and 0.2 μMSYTOX Green. Upon addition of Lysis Solution 1, the tube was immediatelyvortexed (medium setting) for 5 seconds. These samples were kept at roomtemperature for 1 hr. At this time, 1 ml “Lysis Solution 2” was injectedforcefully into each tube, which were immediately vortexed (medium) for5 seconds. Lysis Solution 2 was prepared with deionized water and 85.6mg/ml sucrose, 15 mg/ml citric acid, and 0.2 μM SYTOX Green. Thesespecimens were maintained at room temperature for 30 minutes.Subsequently, samples were stored at 4° C. until flow cytometricanalysis (up to two days following cell harvest/staining/lysisprocedures).

Flow Cytometric Scoring of Micronuclei: Instrumentation and Gating

Samples, stored for up to two days at 4° C., were gently tapped toresuspend the particles. Data acquisition and analysis was thenaccomplished with a single-laser flow cytometer, 488 nm excitation(FACSCalibur, BD Biosciences, San Jose, Calif.). Instrumentationsettings and data acquisition/analysis were controlled with CellQuestsoftware v3.3 (BD Biosciences). SYTOX-associated fluorescence emissionwas collected in the FL1 channel (530/30 band-pass filter), andEMA-associated fluorescence was collected in the FL3 channel (670long-pass filter). Events were triggered on FL1 fluorescence. The FCMgating strategy that was developed for this micronuclei scoringapplication required events to meet each of six separate criteria beforethey were scored as nuclei or micronuclei (see FIGS. 2A-H). Theincidence of flow cytometric-scored micronuclei is expressed asfrequency percent (no. micronuclei/no. nuclei×100), and are based on theacquisition of 20,000 EMA-negative nuclei per specimen.

Microscopy-Based Scoring of Micronuclei

Approximately 3.5×10⁶ cells per culture were centrifuged and resuspendedwith 20 μl heat-inactivated fetal bovine serum. 5 μl aliquots wereapplied to glass slides. Air dried slides were submerged in absolutemethanol for 10 minutes and then stored in the dark until staining andanalysis. Staining was accomplished by submerging slides for 60 secondsin acridine orange solution (12.5 mg/ml, prepared in PBS). An OlympusBH-2 fluorescence microscope was used for micronuclei measurements at400× magnification. For each treated culture, 2000 mononucleated,non-apoptotic, non-necrotic cells were analyzed for the presence ofmicronuclei (1000 cells×2 slides per culture). To be scored as amicronuclei-containing cell, the micronuclei event(s) had to beapproximately round in shape, exhibit similar staining characteristicsas the main nucleus, less than ⅓ the size of the main nucleus, and couldnot overlap with the main nucleus. The incidence of micronucleated cellsis presented as mean frequency percent.

Statistical Analyses

Mean % micronuclei and standard error of the mean (“SEM”) values werecalculated in Excel (Office X for Mac®, Microsoft Corp., Seattle,Wash.). To assess the degree to which mean flow cytometric-based %micronuclei data corresponded to parallel microscopy-based measurements,non-parametric Spearman's coefficients (rs) were calculated for eachchemical studied (JMP Software, v5 for Mac®, SAS Institute, Cary, N.C.).

EXAMPLE 1 Heat Shock Treatment

L5178Y cells were exposed to an elevated temperature (“heat shock”) toevaluate the stage of apoptosis at which EMA staining occurs. Other morecommonly used labeling techniques were studied to provide a frame ofreference. Representative time-course data are presented in FIG. 3. Asexpected, cell surface expression of phosphatidyl serine, asdemonstrated by Annexin V-labeling, occurred most rapidly in response toheat treatment. In contrast, exclusion of the charged nucleic acid dyepropidium iodide was only modestly affected over this time-course.Relative to annexin and propidium iodide, an intermediate rate ofEMA-positive labeling was observed. In fact, EMA performed quitesimilarly to YO-PRO-1, a nucleic acid dye that has been described as arelatively early-stage apoptosis marker (Idziorek et al., “YOPRO-1Permits Cytofluorometric Analysis of Programmed Cell Death (Apoptosis)Without Interfering with Cell Viability,” J. Immunol. Methods185:249-258 (1995), which is hereby incorporated by reference in itsentirety).

A subsequent heat shock experiment evaluated the degree to which EMAstaining, followed by detergent and SYTOX incubation, is able todifferentially label micronuclei and apoptotic bodies. Micronuclei werescored via flow cytometry, with one exception to the standard proceduredescribed above. For this experiment, micronuclei frequencies weredetermined for these preparations both with and without the EMA-staininggate/criterion (see FIG. 2F). As shown by FIG. 4, when flow cytometricanalyses did not exclude EMA-positive events, chromatin from dead/dyingcells was observed to significantly impact the micronuclei scoringregion. Conversely, when EMA-positive particles were excluded from theseanalyses, baseline micronuclei frequencies were observed. These datasupport the hypothesis that EMA may be useful for protecting flowcytometric-based micronuclei counts from spuriously high readings due tothe presence of dead and dying cells.

EXAMPLE 2 Genotoxicants

Each of the six genotoxicants studied was observed to cause adose-related increase in micronucleus frequency (FIGS. 5A-G). Asdemonstrated by the low degree of variation among replicate flowcytometric samples, reproducible % micronuclei values were observed forthe automated scoring process for both vehicle and genotoxicant-treatedcultures. Furthermore, the correspondence between flow cytometric- andmicroscopy-based values was high (r_(s) values ranged from 0.7 to 1.0;see Table 2).

TABLE 2 Overview of Micronucleus Test Results (L5178Y Cells) HighestConc. MN FCM/Microscopy Cell Treatment/Recovery Tested InductionSpearman Cycle Chemical (hrs) (μg/ml) (≧3-fold) Correlation Effect*Methyl 4/20 30 Yes 1.000 +++ methanesulfonate Hydroxyurea 4/20 18 Yes0.9429 +++ Etoposide 4/20 0.1 Yes 1.000 ++ Cyclophosphamide 4/20 6 Yes1.000 + monohydrate Benzo[a]pyrene 4/20 4 Yes 0.9370 − Vinblastinesulfate 4/20 0.3 Yes 0.7000 +++ Vinblastine sulfate 24/0  0.00075 Yes1.000 +++ Tributyltin 4/20 0.15 No 0.9487 − methoxide Tributyltin 24/0 0.06 No 0.4104 − methoxide Dexamethasone 4/20 300 No 0.2868 −Dexamethasone 24/0  100 No −0.4472 − Sucrose 4/20 5000 No 0.2236 −Sucrose 24/0  5000 No 0.3482 − *Qualitative assessment of SYTOXfluorescence histogram (− no difference relative to solvent control; +++distinctly different).

The benzo[a]pyrene graph (FIG. 5E) shows flow cytometric-based solventcontrol % micronuclei values that are moderately elevated relative toparallel microscopy and other flow cytometric-based solvent control datasets. This experiment was performed early in the assay developmentprocess, and these elevated counts are attributable to unoptimized cellprocessing. Specifically, just prior to flow cytometry analysis,nuclei/micronuclei particles were resuspended with a pipettor as opposedto gentle tapping. One likely explanation for these modestly elevatedbaseline counts involves the fate of metaphase chromosomes. The two-steplysis procedure provides mitotic chromosomes which form rather tightlyassociated bundles (confirmed by microscopic inspection). In thisaggregate form, they do not impact the micronuclei scoring region.However, it has been found that at this point in the process, it ispossible to disassociate metaphase chromosomes when specimens arehandled too vigorously. When this occurs, liberated metaphasechromosomes, with their micronucleus-like DNA content, can affectmicronuclei frequency measurements. This explanation is supported byexperiments with colcemid (0-4 hr treatments to accumulate cells inmetaphase). Flow cytometric analysis of these specimens producedbaseline-like micronuclei frequencies, but only when particles areresuspended by gentle tapping as opposed to pipetting or vigorousvortexing.

Given reports that some non-DNA reactive genotoxicants may requireextended treatment durations to maximally express micronuclei(Matsushima et al., “Validation Study of the In Vitro Micronucleus Testin a Chinese Hamster Lung Cell Line (CHL/IU),” Mutagenesis 14:569-580(1999), which is hereby incorporated by reference in its entirety)vinblastine was studied with a 24 hr continuous exposure in addition tothe short-term treatment schedule. As shown by FIGS. 5F-G, thevinblastine-induced micronucleus response was indeed greater given thecontinuous exposure scheme. The discrepancy in micronucleus responseobserved at the highest concentration of this potent aneugen may berelated to the difficulty of scoring slides noted by the microscopist atthis cytotoxic drug level.

EXAMPLE 3 Non-Genotoxicants

As shown in FIG. 5H, sucrose was neither cytotoxic nor genotoxic up to 5mg/ml in the short-term exposure scenario. Based on expert working grouprecommendations, negative short-term results were followed by acontinuous-treatment exposure (Kirsch-Volders et al., “Report from theIn Vitro Micronucleus Assay Working Group,” Mutat. Res. 540:153-163(2003), which is hereby incorporated by reference in its entirety).Again, micronucleus-induction was not evident (see FIG. 5I). Correlationto microscopy is poorly depicted by the r_(s) values that appear inTable 2. This may be explained by the extreme sensitivity of thisstatistic when % micronuclei frequencies are fluctuating in the range ofbaseline values.

The apoptosis-inducing agents tributyltin methoxide and dexamethasonewere also investigated in both short-term and continuous exposurescenarios. Unlike sucrose, these experiments included concentrationswhich significantly affected cell survival (FIGS. 5J-M). Nil to slightchanges in % micronuclei were observed for these chemicals even atcytotoxic concentrations. Both exposure scenarios resulted in less than3-fold increase relative to solvent control. Continuous exposureresulted in fold increases which more closely approached this a prioricutoff. If this becomes a common finding as more non-genotoxicants arestudied, it may be advantageous to explore the sensitivity andspecificity of the assay when the cytotoxicity-based limit concentrationfor long-term treatments is lower than for short-term treatments.Another option may be to use additional criteria for setting the topconcentration (for example % EMA-positive events, discussed in moredetail, infra).

In any event, the tributyltin methoxide and dexamethasone data presentedherein suggest that the EMA/SYTOX sequential staining method provides aneffective layer of protection from spurious events derived from dead anddying cells. The system's relative insensitivity to appreciable levelsof cytotoxicity is in stark contrast to tributyltin methoxide resultsreported by Nüsse et al., “Flow Cytometric Analysis of Micronuclei inCell Cultures and Human Lymphocytes: Advantages and Disadvantages,”Mutat. Res. 392:109-115 (1997), which is hereby incorporated byreference in its entirety, who used this chemical to demonstrate theunreliable nature of FCM-based micronuclei data in the context ofapoptotic cultures.

EXAMPLE 4 Supplemental Information: Cell Cycle

In addition to supplying micronuclei frequency data, it was found thatflow cytometry could also provide concurrent information regarding cellcycle effects. That is, by displaying the SYTOX-associated signal asfluorescence area (FL1-A), qualitative assessments of cell cycle-relatedeffects were possible. These analyses clearly demonstrateddose-dependent perturbations to cell cycle for each of the genotoxicantsstudied (see Table 2). For example, FIGS. 6A-C illustrate the G2/M blockobserved for short-term methyl methanesulfonate treatment. Note thatwhile these observations are presented in a qualitative manner, softwarepackages are available which are capable of deconvoluting suchhistograms to provide quantitative assessment of cell cycle effects.

EXAMPLE 5 Supplemental Information: EMA-Positive Phenotype

In addition to supplying genotoxicity and cell cycle information, theseflow cytometric analyses were capable of concurrently providing ameasure of cytotoxicity. Specifically, the frequency of EMA-positiveevents is a statistic that relates to membrane integrity. For instance,except for non-cytotoxic sucrose, each of the other chemicals studiedwas observed to cause a dose-dependent increase in % EMA-positive events(see FIGS. 7A-D). This statistic could be useful for helping to identifychemical concentrations that are too cytotoxic for reliable genotoxicityassessment.

EXAMPLE 6 Second Cell Line

CHO-K1 cells treated with methyl methanesulfonate exhibited adose-related increase in micronucleus frequency (FIG. 8). Asdemonstrated by the low degree of variation among replicate flowcytometric samples, reproducible % micronuclei values were observed forthe automated scoring process for both vehicle and genotoxicant-treatedcultures. Furthermore, the correspondence between flow cytometric- andmicroscopy-based values was high (r_(s) value=1.0). The morphology andgrowth characteristics of CHO cells are quite different from L5178Y (theformer is an attachment cell line with fibroblast-like appearance, whilethe latter grows as a suspension culture with a lymphoblastoid-likeappearance). Nonetheless, these mammalian cells that exhibit differentmorphologies and that originated from different species were found to becompatible with the cell staining, lysis, and flow cytometric analysisprocedures described herein. Collectively, these data provide strongevidence of a robust scoring system.

CONCLUSIONS

Past concerns regarding the degree to which apoptosis adversely affectsflow cytometry-based measurements were clearly warranted (Viaggi et al.,“Flow Cytometric Analysis of Micronuclei in the CD2+ Subpopulation ofHuman Lymphocytes Enriched by Magnetic Separation,” Int. J. Radiat.Biol. 67:193-202 (1995); Nüsse et al., “Flow Cytometric Analysis ofMicronuclei in Cell Cultures and Human Lymphocytes Advantages andDisadvantages,” Mutat. Res. 392:109-115 (1997), which are herebyincorporated by reference in their entirety). The present datareinforces this contention, and also provides a procedure for minimizingor substantially eliminating this problem. The EMA/SYTOX procedureprovided reproducible flow cytometry-based micronuclei measurements thatcorrespond well to parallel microscopy-based values, even when chemicalswere tested to cytotoxic concentrations. The fact that EMA labelsnecrotic cells as well as apoptotic cells before nuclear fragmentationhas occurred, together with its photoactivation property, are importantcharacteristics that each contributed to the optimization of thislabeling scheme.

This and other automated techniques for scoring in vitro-derivedmicronuclei will likely benefit screening programs sooner thanGLP-compliant tests that are performed to support the registration ofnew chemicals and pharmaceuticals. Even so, data presented hereinsuggest that flow cytometry is a platform that could potentially serveboth purposes. Additional experience with diverse chemicals, especiallythose that are cytotoxic but presumably non-genotoxic, will allow forthorough validation of this system.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed is:
 1. A method for the enumeration of mammalian cellmicronuclei, while distinguishing micronuclei from the chromatin of deadand dying cells, the method comprising: exposing a mammalian cell samplecomprising viable cells and dead and dying cells to (i) first and secondfluorescent DNA dyes that are characterized by fluorescent emissionspectra that do not substantially overlap, and (ii) a lysis solutionthat lyses cellular membranes but retains nuclear membranes, saidexposing being carried out under conditions effective to allow the firstfluorescent DNA dye to label chromatin of dead and dying cells but notviable cells and the second DNA dye to label nuclei and/or micronucleifrom all cells; exciting the first and second fluorescent DNA dyes; anddetecting and distinguishing fluorescent emission and light scatterproduced by the nuclei and/or micronuclei while excluding fluorescentemission by the chromatin of dead and dying cells.
 2. The methodaccording to claim 1 further comprising treating the mammalian cellsample with a chemical or physical agent prior to said exposing.
 3. Themethod according to claim 2, wherein the chemical or physical agentcauses genetic damage, the method further comprising: treating themammalian cell sample with a second agent that may modify genetic damagecaused by the chemical or physical agent.
 4. The method according toclaim 1 further comprising: counting micronuclei and nuclei in thesample to calculate frequency of micronuclei per nuclei in the sample.